scholarly journals An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

2021 ◽  
Author(s):  
Xiaojing Chi ◽  
Xinhui Zhang ◽  
Shengnan Pan ◽  
Yanying Yu ◽  
Tianli Lin ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Neutralizing Antibodies ◽  
Clinical Applications ◽  
Life Extension ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Potential Binding ◽  
Rapid Accumulation ◽  
Heavy Chain Antibody ◽  
Human Igg1

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, natural antibodies isolated from convalescent patients are vulnerable to SARS-CoV-2 Spike mutations. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb dimer, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern or interest. Deep-mutational scanning experiments identify the potential binding epitopes of the monomeric Nb1 and Nb2 on the RBD and demonstrate that bivalent Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that Nb1-Nb2 broadly neutralizes SARS-CoV-2, including variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to the biparatopic Nb (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0*10E-12 M) and neutralizing activity (IC50 = 0.0017 nM). Together, we developed a biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

2021 ◽  
Author(s):  
Carl Graham ◽  
Jeffrey Seow ◽  
Isabella Huettner ◽  
Hataf Khan ◽  
Neophytos Kouphou ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Infectious Virus ◽  
Antigenic Drift ◽  
Host Cells ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

scholarly journals A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

2020 ◽  
Author(s):  
Xiaojian Han ◽  
Yingming Wang ◽  
Shenglong Li ◽  
Chao Hu ◽  
Tingting Li ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Inhibitory Concentration ◽  
Emerging Infectious Diseases ◽  
Receptor Binding Domain ◽  
Screening System ◽  
High Potency ◽  
Preliminary Screening ◽  
Neutralization Activity ◽  
Prevention And Treatment ◽  
Efficient Screening

AbstractNeutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

scholarly journals CoV-RBD121-NP Vaccine Candidate Protects against Symptomatic Disease Following SARS-CoV-2 Challenge in K18-hACE2 Mice and Induces Protective Responses That Prevent COVID-19-Associated Immunopathology

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1346
Author(s):  
Jennifer K. DeMarco ◽  
Joshua M. Royal ◽  
William E. Severson ◽  
Jon D. Gabbard ◽  
Steve Hume ◽  
...  
Keyword(s):  
Antibody Response ◽  
Neutralizing Antibodies ◽  
Potential Role ◽  
Vaccine Candidate ◽  
Severe Disease ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Receptor Binding Domain ◽  
Symptomatic Disease ◽  
Human Igg1

We developed a SARS-CoV-2 vaccine candidate (CoV-RBD121-NP) comprised of a tobacco mosaic virus-like nanoparticle conjugated to the receptor-binding domain of the spike glycoprotein of SARS-CoV-2 fused to a human IgG1 Fc domain. CoV-RBD121-NP elicits strong antibody responses in C57BL/6 mice and is stable for up to 12 months at 2–8 or 22–28 °C. Here, we showed that this vaccine induces a strong neutralizing antibody response in K18-hACE2 mice. Furthermore, we demonstrated that immunization protects mice from virus-associated mortality and symptomatic disease. Our data indicated that a sufficient pre-existing pool of neutralizing antibodies is required to restrict SARS-CoV-2 replication upon exposure and prevent induction of inflammatory mediators associated with severe disease. Finally, we identified a potential role for CXCL5 as a protective cytokine in SARS-CoV-2 infection. Our results suggested that disruption of the CXCL5 and CXCL1/2 axis may be important early components of the inflammatory dysregulation that is characteristic of severe cases of COVID-19.

scholarly journals Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

2021 ◽  
Author(s):  
Micah Rapp ◽  
Yicheng Guo ◽  
Eswar R. Reddem ◽  
Lihong Liu ◽  
Pengfei Wang ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Neutralizing Antibodies ◽  
Structural Basis ◽  
List Type ◽  
Receptor Binding Domain ◽  
Heavy Chains ◽  
Vh Gene ◽  
Antibody Class ◽  
Insight Into ◽  
Potent Neutralization

SUMMARYAntibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilized VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bound both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD – a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions.HighlightsDetermine structures of VH1-2-derived antibodies 2-43, 2-15, and H4 in complex with SARS-CoV-2 spikeDefine a multi-donor VH1-2-antibody class with modular components for RBD and quaternary recognitionReveal structural basis of RBD-up and RBD-down recognition within the classShow somatic hypermutations and avidity to be critical for potencyDelineate changes in spike conformation induced by CDRH3-mediated quaternary recognition

scholarly journals A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

2021 ◽  
Vol 17 (3) ◽  
pp. e1009328
Author(s):  
Hebang Yao ◽  
Hongmin Cai ◽  
Tingting Li ◽  
Bingjie Zhou ◽  
Wenming Qin ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Motif ◽  
Fusion Partner ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Neutralization Activity ◽  
Effective Strategies ◽  
High Affinity ◽  
Considerable Research

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

scholarly journals Decline of humoral responses against SARS-CoV-2 Spike in convalescent individuals

2020 ◽  
Author(s):  
Guillaume Beaudoin-Bussières ◽  
Annemarie Laumaea ◽  
Sai Priya Anand ◽  
Jérémie Prévost ◽  
Romain Gasser ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Positive Role ◽  
Neutralization Activity ◽  
Specific Igg ◽  
Humoral Responses ◽  
Over Time

ABSTRACTIn the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to COVID-19 patients. The therapy has been deemed safe and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here we performed repeated analyses at one-month interval on 31 convalescent individuals to evaluate how the humoral responses against the SARS-CoV-2 Spike, including neutralization, evolve over time. We observed that receptor-binding domain (RBD)-specific IgG slightly decreased between six and ten weeks after symptoms onset but RBD-specific IgM decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing SARS-CoV-2 S wild-type or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after symptoms resolution.

scholarly journals Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population

2020 ◽  
Author(s):  
Zhigang Yi ◽  
Yun Ling ◽  
Xiaonan Zhang ◽  
Jieliang Chen ◽  
Kongying Hu ◽  
...  
Keyword(s):  
Viral Infection ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Neutralization Assay ◽  
Receptor Binding Domain ◽  
E Proteins ◽  
Neutralization Activity ◽  
Vaccine Candidates ◽  
Linear Epitopes ◽  
Specific Antigens

Pandemic SARS-CoV-2 has infected over 10 million people and caused over 500,000 mortalities. Vaccine development is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with their elicited neutralizing antibodies in the convalescent COVID-19 population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent serums and 24 non-COVID-19 serums. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent serums. Epitopes in the receptor-binding domain (RBD) of S ill reacted with the convalescent serums. Of note, two peptides non-specifically interacted with most of the non-COVID-19 serums. Neutralization assay indicated that only five serums completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent serums, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.

scholarly journals Decline of Humoral Responses against SARS-CoV-2 Spike in Convalescent Individuals

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Guillaume Beaudoin-Bussières ◽  
Annemarie Laumaea ◽  
Sai Priya Anand ◽  
Jérémie Prévost ◽  
Romain Gasser ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Active Infection ◽  
Positive Role ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Specific Igg ◽  
Alternative Approaches ◽  
Humoral Responses

ABSTRACT In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease 2019 (COVID-19) patients. The therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here, we performed repeated analyses at 1-month intervals on 31 convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein, including neutralization, evolve over time. We observed that the levels of receptor-binding-domain (RBD)-specific IgG and IgA slightly decreased between 6 and 10 weeks after the onset of symptoms but that RBD-specific IgM levels decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type SARS-CoV-2 S or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms. IMPORTANCE While waiting for an efficient vaccine to protect against SARS-CoV-2 infection, alternative approaches to treat or prevent acute COVID-19 are urgently needed. Transfusion of convalescent plasma to treat COVID-19 patients is currently being explored; neutralizing activity in convalescent plasma is thought to play a central role in the efficacy of this treatment. Here, we observed that plasma neutralization activity decreased a few weeks after the onset of the symptoms. If neutralizing activity is required for the efficacy of convalescent plasma transfer, our results suggest that convalescent plasma should be recovered rapidly after the donor recovers from active infection.

scholarly journals Persistence of robust humoral immune response in COVID-19 convalescent individuals over 12 months after infection

2021 ◽  
Author(s):  
Kei Miyakawa ◽  
Sousuke Kubo ◽  
Sundararaj Stanleyraj Jeremiah ◽  
Hirofumi Go ◽  
Yutaro Yamaoka ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Virus Antibody ◽  
Neutralization Activity ◽  
Humoral Immune ◽  
Antibody Titers ◽  
Viral Nucleocapsid

SARS-CoV-2 infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). Here we report the persistence of nAb responses over 12 months after infection despite its decreasing trend noticed from 6 months. The study included sera from 358 individuals who had been infected with SARS-CoV-2 between January and May 2020. Samples were collected at 6 and 12 months after onset. The titers of IgG to the viral nucleocapsid protein (NP) and receptor-binding domain of the spike protein (RBD) were measured by CLEIA. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. While the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases (~30%) was undermined, implying the susceptibility of reinfection to the variants of concerns (VOCs). COVID-19 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

scholarly journals A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

2020 ◽  
Author(s):  
Hebang Yao ◽  
Hongmin Cai ◽  
Tingting Li ◽  
Bingjie Zhou ◽  
Wenming Qin ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Motif ◽  
Fusion Partner ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Neutralization Activity ◽  
Effective Strategies ◽  
High Affinity ◽  
Considerable Research

ABSTRACTA key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

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